Publisher Summary Studies of cytotoxicity by human lymphocytes revealed not only that both allogeneic and syngeneic tumor cells were lysed in a non-MHC-restricted fashion, but also that lymphocytes from normal donors were often cytotoxic. Lymphocytes from any healthy donor, as well as peripheral blood and spleen lymphocytes from several experimental animals, in the absence of known or deliberate sensitization, were found to be spontaneously cytotoxic in vitro for some normal fresh cells, most cultured cell lines, immature hematopoietic cells, and tumor cells. This type of nonadaptive, non-MHC-restricted cellmediated cytotoxicity was defined as “natural” cytotoxicity, and the effector cells mediating natural cytotoxicity were functionally defined as natural killer (NK) cells. The existence of NK cells has prompted a reinterpretation of both the studies of specific cytotoxicity against spontaneous human tumors and the theory of immune surveillance, at least in its most restrictive interpretation. Unlike cytotoxic T cells, NK cells cannot be demonstrated to have clonally distributed specificity, restriction for MHC products at the target cell surface, or immunological memory. NK cells cannot yet be formally assigned to a single lineage based on the definitive identification of a stem cell, a distinct anatomical location of maturation, or unique genotypic rearrangements.

Biology of natural killer cells.

Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs -- also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-alpha) reprogram macrophages by releasing prostaglandin E(2) that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups.

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Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production.

Natural killer cells are a recently discovered subpopulation of lymphoid cells that are present in most normal individuals of a range of mammalian and avian species. Natural killer cells have spontaneous cytolytic activity against a variety of tumor cells and some normal cells, and their reactivity can be rapidly augmented by interferon. They have characteristics distinct from other types of lymphoid cells and are closely associated with large granular lymphocytes, which comprise about 5 percent of blood or splenic leukocytes. There is increasing evidence that natural killer cells, with the ability to mediate natural resistance against tumors in vivo, certain virus and other microbial diseases, and bone marrow transplants, may play an important role in immune surveillance.

https://science.sciencemag.org/content/sci/214/4516/24.full.pdf

Natural killer cells: their roles in defenses against disease

Three independent variants with a profound reduction of cell surface H-2 have been selected from the C57BL/6 mouse-derived RBL-5 and EL-4 T lymphomas. After subcutaneous inoculation of low cell doses in syngeneic mice, the H-2- variants failed to grow out, whereas the H-2+ control lines showed progressive growth. No difference in growth rate or cloning efficiency was detectable in tissue culture. The in vivo difference in tumor outgrowth was analyzed in detail for one of the H-2-low lines. The outgrowth difference remained after the H-2-low variant and the control line had been injected subcutaneously in opposite flanks of the same mouse, and it was not dependent upon activity of mature T cells, since the same result was seen in athymic nude mice. The difference was partially sensitive to irradiation of the hosts. When mice were pretreated with anti-asialo GM1 antiserum, known to depress natural killer (NK) cell activity, the difference in outgrowth was abolished, and both the control line and the H-2- variant showed progressive growth in vivo. Experiments comparing the distribution and survival of isotope-prelabeled variant and wild type cells indicated that a rapid elimination of the former took place within 24 h after intravenous injection. These differences in tumor elimination were not seen in mice treated with anti-asialo GM1 antiserum. We conclude that the reduced tumorigenicity of sublines with impaired H-2 expression is largely, if not exclusively due to rapid elimination by NK cells. These findings may reflect an inverse, indirect relation between factors controlling H-2 expression and NK sensitivity. Another possible explanation is that major histocompatibility complex (MHC)-encoded gene products are directly involved in a regulatory signal in the NK cell system. According to this interpretation, immunological selectivity in the NK cell system would be achieved by the failure to recognize self-MHC, irrespective of the presence of foreign antigens, i.e. by detection of no-self rather than of nonself. This may also explain previous observations on H-2-linked hybrid resistance against lymphoid grafts and changes in H-2 phenotypes associated with tumor progression.

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Host resistance directed selectively against H-2-deficient lymphoma variants. Analysis of the mechanism.

Animal models, by definition, are an approximation of reality, and their use in developing anti-cancer drugs is controversial. Positive retrospective clinical correlations have been identified with several animal models, in addition to limitations and a need for improvement. Model inadequacies include experimental designs that do not incorporate biological concepts, drug pharmacology, or toxicity. Ascites models have been found to identify drugs active against rapidly dividing tumors; however, neither ascitic nor transplantable subcutaneous tumors are predictive of activity for solid tumors. In contrast, primary human tumor xenografts have identified responsive tumor histiotypes if relevant pharmacodynamic and toxicological parameters were considered. Murine toxicology studies are also fundamental because they identify safe starting doses for phase I protocols. We recommend that future studies incorporate orthotopic and spontaneous metastasis models (syngeneic and xenogenic) because they incorporate microenvironmental interactions, in addition to confirmatory autochthonous models and/or genetically engineered models, for molecular therapeutics. Collectively, murine models are critical in drug development, but require a rational and hierarchical approach beginning with toxicology and pharmacology studies, progressing to human primary tumors to identify therapeutic targets and models of metastatic disease from resected orthotopic, primary tumors to compare drugs using rigorous, clinically relevant outcome parameters.

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Murine models to evaluate novel and conventional therapeutic strategies for cancer.

Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid "asialo GM1" was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti-asialo GM1 antiserum showed little cross-reactivity with structurally related glycolipids, e.g. GM), GD 1 b and asialo GM2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti-NK activity with graded amounts of asialo GM1 but not with other glycosphingolipids. The absorption of anti-brain-associated T cell antigen (anti-BAT) with asialo GM1 also effectively diminished its anti-NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM1 is responsible for the anti-NK activity contained in the anti-BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti-asialo GM1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti-asialo GM1 and complement. These results indicate that asialo GM1 is expressed on mouse NK cells in a high concentration.

A glycolipid on the surface of mouse natural killer cells

Intravenous injection of anti-asialo GM1, which has been shown to eliminate natural killer (NK) activity in vitro in the presence of complement, completely abolished NK activity against lymphoma cell line (YAC-1) in spleen cells from athymic nude mice as well as from conventional mice. An immunofluorescence study revealed a decreased number of asialo GM1 positive cells in the spleens of mice injected with anti-asialo GM1 than in those of mice injected with normal rabbit serum. In the nude mice with reduced NK activity, incidence of tumor take and the growth were enhanced when syngeneic (RL male-1), and allogeneic (YAC-1) tumors and human tumors were transplanted subcutaneously. These results strongly suggest that NK cells play an important role in transplanted-tumor growth in vivo.

In vivo effects of anti-asialo GM1. I. Reduction of NK activity and enhancement of transplanted tumor growth in nude mice.

A high incidence of autoantibody against the neutral glycolipid "asialo GM1" was observed in sera from patients with systemic lupus erythematosus (SLE) with neurological disorders, using an immunoflocculation test. The sera from 14 out of 17 cases of SLE with neurological disorders showed antibody activity against asialo GM1 but not against the following glycolipids: asialo GM2 GM1, and galactocerebroside. In another 87 cases of SLE without any history of seizures, as well as 61 cases of other autoimmune diseases (rheumatoid arthritis, progressive systemic sclerosis, mixed connective tissue disease, etc.) and 20 cases of various neurological diseases (epilepsy, multiple sclerosis, etc.), no antibody could be detected. In general, the antibody titer was high several months, even years, before and/or after the seizure, though the titer was low at the time that patients showed definite neurological symptoms. Immunochemical characterization with Sephadex G-200 chromatogrphy and protein A-Sepharose CL-4B affinity column indicated that the antiasialo GM1 was probably an autoantibody belonging to the immunoglobulin G class. The above results suggest that this newly found autoantibody plays a role in the pathogenesis of neurological disorders accompanying SLE.

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Antiglycolipid autoantibody detected in the sera from systemic lupus erythematosus patients.

Antibody directed to the neutral glycolipid "asialo GM1" was found to react with the majority of thymus lymphoid cells lacking characteristic T cell markers in mice at an early embryonic stage (13 days of gestation). The proportion of these asialo GM1-positive cells (asialo GM1+ cells) decreased strikingly thereafter, contrasting with an increased fraction of lymphocytes possessing T cell surface markers such as Thy-1, Lyt-1, and Lyt-2. After about 18 gestational days, only a few asialo GM1+ cells were detected in the thymus as well as in other lymphoid organs. Double-staining analysis of the embryonic thymocytes (13 days of gestation) with anti-asialo GM1 and anti-Thy-1 demonstrated that thymocytes stained with anti-asialo GM1 did not react with anti-Thy-1, and vice versa. Morphologic examination by immunoelectronmicroscopy demonstrated that these asialo GM1+ cells were mainly composed of immature, large lymphoid cells having large nucleoli and relatively abundant cytoplasm with many polysomes. These results suggest that asialo GM1 is present on very early thymocytes and is lost as the mature murine T cell protein antigens Thy-1, Lyt-1, and Lyt-2 develop on these cells. The antibody to this glycolipid is a useful tool for studying embryonic thymic differentiation.

Y Nagai

Papers

A glycolipid on the surface of mouse natural killer cells

In vivo effects of anti-asialo GM1. I. Reduction of NK activity and enhancement of transplanted tumor growth in nude mice.

Antiglycolipid autoantibody detected in the sera from systemic lupus erythematosus patients.

The glycolipid asialo GM1 as a new differentiation antigen of fetal thymocytes.